Mass spectrometry works very well for the analysis of most substances. The technique produces very specific data and is very sensitive. However, for the analysis of very polar compounds or high molecular weight substances, mass spectral analyses require special procedures. Probe distillation of pure compounds is the method most often selected. But, probe distillation of pure compounds can only be used if the substance to be analyzed can, at some temperature below its thermal degradation point, be sublimed into the ion source of the mass spectrometer.
In order to circumvent the problems associated with the heating of very polar compounds which can easily decompose with heat, other ionization techniques have been devised for mass spectral analysis. These include field desorption, field ionization, surface ionization and fast atom bombardment. These techniques are usually applied to relatively pure substances and are seldom used for the real-time analysis of polar compounds in complex mixtures. Therefore, a method was needed which would allow first, for the separation of polar compounds and secondly, for the delivery of relatively pure substances into the ion source of the mass spectrometer.
Analytical methods currently employed in many laboratories require samples to be first analyzed by HPLC in one laboratory and then hand carried to the mass spectrometry facility for further analysis. This requires time and additional sample preparation.
However, methods are currently available which allow effluents from high performance liquid chromatography columns to be carried directly into the ion source of the mass spectrometer. These methods can be highlighted into four general groups. Each has its own advantages and associated problems.
1. Direct Introduction: This technique relies on a powerful vacuum pumping system which allows the HPLC effluent to flow directly into the ion source of the mass spectrometer. Very significant increases in vacuum pressure, which is one of the major drawbacks in this technique, are controllable, if only a very small sample of the effluent is presented (split) from the bulk of the HPLC effluent. The direct introduction of HPLC effluent into the ion source does not allow for high resolution mass spectral analyses, which requires very low pressures in the analyzer section and a very stable vacuum. Fluctuations in the vacuum pressure affect the results significantly. Only volatile HPLC solvents can be used with this technique. Additionally, the method is less sensitive because a major portion of the sample is not analyzed. Only chemical ionization appears to be susceptible to analysis by this technique.
2. Thermospray: This technique has become very popular and relies on the rapid evaporation of the HPLC solvent as it passes through a heated capillary tube. A supersonic jet of vapor containing the compounds to be analyzed is allowed to pass adjacent to a draw-out plate which directs ionized compounds directly into the analyzer section of the mass spectrometer. This technique requires the HPLC solvent to be volatile. More significant is the requirement that the ion source of the mass spectrometer needs to be replaced or significantly modified. Thermospraying is not applicable to high resolution mass spectral analysis.
3. Atmospheric Pressure Mass Spectrometry: This technique relies on the use of a nebulizer which mixes the HPLC effluent with a high pressure stream of nitrogen gas. The vaporized HPLC solvent is then allowed to pass through a corona discharge and into the analyzer section of the mass spectrometer. This is a dedicated detector for the HPLC-mass spectrometer. This technique appears to work well for only for more volatile compounds and the best information is obtained only with special (triple quadrupole) analyzer instruments.
4. Membrane Separator: This technique relies on the selective migration of compounds through a porous membrane. The membrane is placed in front of the ion source and the effluent of the HPLC column is allowed to pass over this porous material. This type of HPLC-MS interface allows substantial amounts of the HPLC solvent into the mass spectrometer. The temperature of the mass spectrometer, flow rates and type of membrane material can greatly affect the migration of the compounds through the porous probe tip and thus significantly affect the resolution and sensitivity of the analyses.
5. Moving Belt Interface: This technique relies on the deposition of the HPLC solvent containing the sample to be analyzed, onto a moving wire or ribbon. The HPLC solvent is allowed to evaporate (by vacuum pumping and/or heating) directly off the moving belt as it is deposited thereon as the belt passes under the exit port of the HPLC column. The sample residue from the evaporation step is then carried directly into the ion source of the mass spectrometer. As the moving belt enters the ion source, chemicals are either vaporized off the surface of the belt by the application of external heat or ionized directly on the surface by bombardment with an electron beam. The integrity of the vacuum of the mass spectrometer is maintained substantially undisturbed, because the HPLC solvents are removed a considerable distance from the ion source and thus do not enter the ion source.
The moving belt interface is more amenable to high resolution mass spectral analysis. The ion source, analyzer section and the vacuum pumping section of the mass spectrometer do not necessarily need to be significantly altered or replaced, although commercially available interfaces seem to require that the interface must be permanently bolted to the normal solid probe inlet system of the mass spectrometer. However, the prior art moving belt interface has its own drawbacks. Because of the time delay between the deposition of the sample on the moving belt and analysis as the belt moves into the ion source of the mass spectrometer (20-30 seconds), volatile compounds can be lost in the vacuum system of the interface. As with the other techniques herein described, the moving belt interface can not handle HPLC solvents which contain nonvolatile buffer components. The mode and timing of the introduction of the HPLC effluent into the HPLC-MS interface is most critical. An uneven application can result in an overlap of compounds which were completely resolved by the HPLC column.
Various types of approaches to improving moving belt interfaces have been previously considered. These prior art approaches are exemplified by the following publications and patents:
1. "Deposition Method For Moving Ribbon Liquid Chromatograph-Mass Spectrometer Interfaces", R. D. Smith et al., Anal. Chem., 53, 739-740 (1981);
2. "Liquid Chromatography-Mass Spectrometry with Electron Impact And Fast Ion Bombardment with A Ribbon Storage Interface", R. D. Smith et al., Anal. Chem., 53, 1603-1611 (1981);
3. "Moving Belt Interface with Spray Deposition For Liquid Chromatography/Mass Spectrometry", M. J. Hayes et al., Anal. Chem., 55, 1745-1752 (1983);
4. "A Comparison Of Moving Belt Interfaces For Liquid Chromatography-Mass Spectrometry", D. C. Games, et al., Biomedical Mass Spectrometry, 11(2), 87-95 (1984);
5. "Simplified Moving Belt Interface For Liquid Chromatography/Mass Spectrometry", S. D. Stout et al., Anal. Chem., 57, 1783-1786 (1985);
6. "Method For The Analysis Of Materials By Chromatography And Mass Spectrometry", U.S. Pat. No. 4,501,817 issued Feb. 26, 1985, to Brian D. Andresen and Kwokei J. Ng;
7. "Liquid Chromatograph/Mass Spectrometer Interface", U.S. Pat. No. 4,160,161 issued July 3, 1979 to Robert L. Horton;
8. "Interface For Use In A Combined Liquid Chromatography-Mass Spectrometry System", U.S. Pat. No. 4,112,297 issued Sept. 5, 1978 to Hirayuki Miyogi et al;
9. "Liquid Cnromatograph/Mass Spectrometer Interface", U.S. Pat. No. 4,055,987 issued Nov. 1, 1977 to william H. McFadden, describes a liquid chromatograph/mass spectrometer interface which permits continuous introduction of dilute solutions into the ion source of a mass spectrometer. A ribbon loop and a drive therefor serve to receive the solution from the liquid chromatograph. The solution is then carried by the moving ribbon or belt into a vacuum chamber and then into the ion source of the mass spectrometer. A heater positioned at or near the position where the solution from the chromatographic solution is received on the moving ribbon and another one placed close or adjacent to the ion source vaporize the solution into the ionization chamber;
10. "Liquid Chromatography-Mass Spectrometry System And Method", U.S. Pat. No. 3,997,298 issued Dec. 14, 1976 to Fred w. McLafferty et al;
11. "Method Of Coupling Thin Layer Cnromatograph with Mass Spectrometer", U.S. Pat. No. 3,896,661 Issued July 29, 1975 to Robert M. Parkhurst et al.
12. "Continuous-Flow Solution Concentrator And Liquid Chromatograph/Mass Spectrometer Interface And Methods For Using Both", U.S. Pat. No. 4,281,246 issued July 28, 1981 to V. Edward white et al describes an interface between a liquid chromatograph and mass spectrometer for conducting a liquid stream from the chromatographic column to the mass spectrometer. The stream passes continuously from the chromatographic column, heated along the way and fed into the ion source of the mass spectrometer; and
13. "Method And Apparatus For The Mass Spectrometric Analysis Of Solutions", U.S. Pat. No. 4,531,056 Issued July 23, 1985 to Michael J. Labowsky et al discloses an electrospray ion source for a mass spectrometer capable of generating ions, which are then pumped into the mass spectrometer through two vacuum chambers.
The major problems with the prior art liquid chromatography/mass spectrometry interfaces are (1) that each of the above mentioned methods for the analysis of HPLC effluent require either a new mass spectrometer which would accept the particular interface design, or (2) that the ion source, vacuum system and other components of the existing mass spectrometer require substantial modifications to accept and function with the interface, or (3) that the method is incapable of analyzing a wide variety of unique compounds which would be contained in the HPLC effluent. No one approach appears to be more desirable than the other and none of them provide a method or means for the direct, real-time analysis of polar compounds in HPLC effluents. Furthermore, these prior art devices are not stand-alone, portable units which can be used with any existing mass spectrometers, or units which are capable of handling the transition between the high pressures of HPLC columns to the near vacuum conditions of the mass spectrometer.
Therefore, a need exists to provide an interface which would allow chemical and biological compounds, particularly polar compounds, to be separated by liquid chromatography (LC), particularly, high performance liquid chromatography (HPLC) and sequentially or simultaneously analyzed by a mass spectrometer (MS). The interface must be capable of analyzing these compounds in real time, stripping away the LC or HPLC solvent as it emerges from the end of the column and leaving a residue suitable for mass spectral analysis. In addition, the interface must retain or maintain an adequate vacuum that is compatible with that in the mass spectrometer. A moving belt device which is capable of stripping away solvents from the LC or HPLC effluents must be provided. Compounds deposited on the belt should be capable of being vaporized or ionized off the surface of the belt (with a heater in the probe tip for example,) or ionized directly by fast atom or electron bombardment or by direct electron impact (surface ionization and such other techniques known in the art of mass spectrometry.
Therefore, it is an object of this invention to provide an improved moving belt interface for real-time LC or HPLC-MS analysis of chemical and biological compounds.
A further object of the invention is to provide an interface which allows polar compounds to be separated by conventional liquid chromatography or high performance liquid chromatography and sequentially or simultaneously analyzed by a mass spectrometer.
Another object of the invention is to provide an HPLC-MS interface which would strip away the LC or HPLC solvent as it emerges from the end of the LC or HPLC column and leave a residue suitable for mass spectral analysis.
Yet another object of the invention is to provide a moving belt interface for an HPLC-MS with a magnetically coupled drive train, a calibrated HPLC delivery system and a heated probe tip.
Another object of the invention is to provide such a moving belt interface which additionally includes a plurality of separate vacuum locks through which the moving belt passes, a portable stand-alone vacuum station, and means located adjacent the probe tip for ionization of the material on the moving belt.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.